Rescue of placental phenotype in a mechanistic model of Beckwith-Wiedemann syndrome.

Abstract

Several imprinted genes have been implicated in the process of placentation. The distal region of mouse chromosome 7 (Chr 7) contains at least ten imprinted genes, several of which are expressed from the maternal homologue in the placenta. The corresponding paternal alleles of these genes are silenced in cis by an incompletely understood mechanism involving the formation of a repressive nuclear compartment mediated by the long non-coding RNA Kcnq1ot1 initiated from imprinting centre 2 (IC2). However, it is unknown whether some maternally expressed genes are silenced on the paternal homologue via a Kcnq1ot1-independent mechanism. We have previously reported that maternal inheritance of a large truncation of Chr7 encompassing the entire IC2-regulated domain (DelTel7 allele) leads to embryonic lethality at mid-gestation accompanied by severe placental abnormalities. Kcnq1ot1 expression can be abolished on the paternal chromosome by deleting IC2 (IC2KO allele). When the IC2KO mutation is paternally inherited, epigenetic silencing is lost in the region and the DelTel7 lethality is rescued in compound heterozygotes, leading to viable DelTel7/IC2KO mice.
Considering the important functions of several IC2-regulated genes in placentation, we set out to determine whether these DelTel7/IC2KO rescued conceptuses develop normal placentae. We report no abnormalities with respect to the architecture and vasculature of the DelTel7/IC2KO rescued placentae. Imprinted expression of several of the IC2-regulated genes critical to placentation is also faithfully recapitulated in DelTel7/IC2KO placentae.
Taken together, our results demonstrate that all the distal chromosome 7 imprinted genes implicated in placental function are silenced by IC2 and Kcnq1ot1 on the paternal allele. Furthermore, our results demonstrate that the methylated maternal IC2 is not required for the regulation of nearby genes. The results show the potential for fully rescuing LQ trans placental abnormalities that are caused by imprinting defects.

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Oh-McGinnis et al. BMC Developmental Biology 2010, 10:50
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Open Access
RESEARCH ARTICLE
Rescue of placental phenotype in a mechanistic
model of Beckwith-Wiedemann syndrome
© 2010 Oh-McGinnis et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Com-
mons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduc-
tion in any medium, provided the original work is properly cited.
Research article
Rosemary Oh-McGinnis1, Aaron B Bogutz1, Kang Yun Lee1, Michael J Higgins2 and Louis Lefebvre*1
Abstract
Background: Several imprinted genes have been implicated in the process of placentation. The distal region of mouse
chromosome 7 (Chr 7) contains at least ten imprinted genes, several of which are expressed from the maternal
homologue in the placenta. The corresponding paternal alleles of these genes are silenced in cis by an incompletely
understood mechanism involving the formation of a repressive nuclear compartment mediated by the long non-
coding RNA Kcnq1ot1 initiated from imprinting centre 2 (IC2). However, it is unknown whether some maternally
expressed genes are silenced on the paternal homologue via a Kcnq1ot1-independent mechanism. We have previously
reported that maternal inheritance of a large truncation of Chr7 encompassing the entire IC2-regulated domain
(DelTel7 allele) leads to embryonic lethality at mid-gestation accompanied by severe placental abnormalities. Kcnq1ot1
expression can be abolished on the paternal chromosome by deleting IC2 (IC2KO allele). When the IC2KO mutation is
paternally inherited, epigenetic silencing is lost in the region and the DelTel7 lethality is rescued in compound
heterozygotes, leading to viable DelTel7/IC2KO mice.
Results: Considering the important functions of several IC2-regulated genes in placentation, we set out to determine
whether these DelTel7/IC2KO rescued conceptuses develop normal placentae. We report no abnormalities with
respect to the architecture and vasculature of the DelTel7/IC2KO rescued placentae. Imprinted expression of several of
the IC2-regulated genes critical to placentation is also faithfully recapitulated in DelTel7/IC2KO placentae.
Conclusion: Taken together, our results demonstrate that all the distal chromosome 7 imprinted genes implicated in
placental function are silenced by IC2 and Kcnq1ot1 on the paternal allele. Furthermore, our results demonstrate that
the methylated maternal IC2 is not required for the regulation of nearby genes. The results show the potential for fully
rescuing trans placental abnormalities that are caused by imprinting defects.
Background
Genomic imprinting is the mechanism by which haploid
maternal and paternal genomes carry different epigenetic
marks, resulting in monoallelic transcription of a subset
of genes which are expressed exclusively from either the
maternal or paternal allele [1]. To date, over eighty
imprinted genes have been identified in humans and mice
[2]. Many of these genes are known to have critical roles
in embryonic development and placentation [3] and have
also been implicated in the regulation of postnatal behav-
iour [4-6]. The imprinted region on distal mouse chro-
mosome 7 (Chr 7) shares syntenic homology with human
chromosome 11p15.5, a region associated with Beckwith-
Wiedemann syndrome (BWS) and Wilms tumor. BWS is
an imprinted disorder that is commonly characterized by
macroglossia, organomegaly, abdominal wall defects,
unusual facial features, hemihypertrophy, hypoglycemia,
exomphalos, ear and renal anomalies [7]. Roughly 5-10%
of BWS patients are predisposed to a variety of childhood
tumours, including hepatoblastoma, rhabdomyosarcoma,
neuroblastoma, adrenal carcinoma, with Wilms tumor
occurring most frequently [7,8].
Distal mouse Chr7 contains several key imprinted genes
required for fetal development, two of which are also
implicated in the etiology of BWS, the paternally
expressed insulin-like growth factor-2 (Igf2) gene regu-
lated by imprinting centre 1 (IC1, also known as H19
DMR) located upstream of the maternally expressed H19
gene and the maternally expressed cyclin-dependent
kinase inhibitor 1C (Cdkn1c) which is regulated by
* Correspondence: louis.lefebvre@ubc.ca
1 Department of Medical Genetics, Molecular Epigenetics Group, Life Sciences
Institute, University of British Columbia, Vancouver, Canada
Full list of author information is available at the end of the article

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imprinting centre 2 (IC2, also known as KvDMR1)
located within intron 10 of the Kcnq1 gene [9,10]. In addi-
tion to Cdkn1c, the IC2 cluster contains at least eight
other maternally expressed genes (Ascl2, Cd81, Tssc4,
Kcnq1, Slc22a18, Phlda2, Osbpl5, and Dhcr7) [11-19]. Of
these, Ascl2, Cd81, and Osbpl5 have been shown to be
expressed and exclusively imprinted in the placenta
[20,21]. The precise functions, if any, of Cd81, Osbpl5,
Tssc4, Kcnq1 and Slc22a18 in the placenta remain to be
elucidated. However, the remaining genes in the cluster
(Ascl2, Phlda2, and Cdkn1c) have well-documented roles
in placentation since knockouts of each of these genes
result in drastic placental phenotypes. Ascl2 null mice fail
to form a spongiotrophoblast layer, a defect leading to
embryonic lethality at E10 [15,22]; Phlda2 null mice are
viable but show an expanded spongiotrophoblast and pla-
centomegaly [23]; Cdkn1c mutants possess varying phe-
notypes including a reduced labyrinth layer, expanded
spongiotrophoblast, placentomegaly and perinatal lethal-
ity [24-26].
The mechanism of silencing of several genes both centro-
meric and distal to the position of the paternal IC2 over a
large domain is not completely understood, though pro-
duction and elongation of the ncRNA Kcnq1ot1 is
thought to play a critical role in this process [27,28].
Kcnq1ot1 production is accompanied by recruitment of
Polycomb group complexes [29] and acquisition of
repressive histone modifications [20,21]. These observa-
tions suggest similarities with the action of other ncRNAs
such as the regulation of Igf2r by the ncRNA Airn [30]
and X-inactivation by Xist [31]. The deletion of the pater-
nal IC2 leads to activation of IC2-regulated genes on the
paternal chromosome and +/IC2KO mice possess a
growth restriction phenotype at least in part because of
biallelic expression of Cdkn1c [32,33]. The placentae of
these animals were also reported to be smaller than their
wild type litter mates [34]. Recent studies have also
shown that IC2 contains CTCF-binding sites which are
occupied only on the unmethylated paternal allele how-
ever it is still unclear what role these CTCF binding sites
might play in silencing the paternal alleles of IC2-regu-
lated genes [35]. It is also not known whether the methy-
lated maternal IC2 allele is important for the activation of
nearby genes.
We previously described an engineered truncation of dis-
tal Chr 7 with a breakpoint upstream of the Ins2 gene
[36]. The resultant 2.7-Mb deletion, called DelTel7,
removes the entire IC2-regulated domain along with the
telomeric end of the Chr 7 domain, replacing it with an
artificial telomere. When the DelTel7 allele is paternally
inherited (+/DelTel7 hemizygotes), there is no obvious
phenotype, showing absence of paternally expressed
imprinted genes required for development or of a haplo-
insufficiency effect [36]. Maternal transmission of
DelTel7 (DelTel7/+ hemizygotes) however, leads to a
mid-gestational lethality, consistent with the deletion of
all maternally expressed genes in the IC2-regulated sub-
domain, and is also accompanied by abnormal placenta-
tion including the lack of spongiotrophoblast and an
expanded trophoblast giant cell layer [36]. Despite a dif-
ference in phenotypic outcome, possibly attributable to
the absence of imprinting of essential genes such as
ASCL2 in humans [37,38], maternal inheritance of
DelTel7 provides a mechanistic model for BWS, which is
often associated with loss of expression of maternally
expressed genes [9]. Remarkably, we also demonstrated
that the lethality observed upon maternal inheritance of
this truncation can be rescued in trans by a paternally
inherited IC2KO allele, which was previously shown to
abolish Kcnq1ot1 transcription and its associated epige-
netic silencing [33]. The compound heterozygous
DelTel7/IC2KO pups survive to term but it is unclear
whether any placental abnormalities or defects in placen-
tal imprinted gene expression remain in the double
mutants. Because of the importance of several IC2-regu-
lated genes in placental function and development, we
present here a characterization of the rescued DelTel7/
IC2KO placentae to determine whether they are compa-
rable to their wild type litter mates and whether deletion
of IC2 leads to a full rescue of the placental phenotype
caused by the loss of maternal genes from the DelTel7
allele. Our assessment would have direct implications for
BWS patients, since placental abnormalities usually
accompany BWS fetuses [39-41]. By examining the archi-
tecture, vasculature, and imprinted gene expression of
the rescued DelTel7/IC2KO placentae we sought to
determine whether it is possible to correct imprinting
defects by restoring gene expression for a large number of
genes in trans and thereby to further elucidate mecha-
nisms of IC2-mediated epigenetic silencing.
Results
Loss of imprinted gene expression in DelTel7/+ placentae
The IC2 imprinted domain of distal Chr 7 contains at
least several maternally expressed mRNA genes and
spans almost 800 kb. The entire domain is deleted in the
DelTel7 allele, a truncation of Chr7 with a breakpoint
upstream of Ins2 and deleting the last 2.7 Mb of Chr7
(Figure 1A). We previously showed that maternal trans-
mission of DelTel7 leads to abnormal placentation and
embryonic lethality at E10.5, whereas paternal hemizy-
gotes are viable [36]. We show here by immunohis-
tochemistry for PHLDA2 (Figure 1B) as well as
quantitative RT-PCR (qRT-PCR) for the imprinted genes
Phlda2, Ascl2, and Cdkn1c, that at E9.5 DelTel7/+ mater-
nal hemizygous placentae are deficient for expression of
these IC2-regulated genes. Placentae were assessed at
E9.5, before the embryonic lethality of the DelTel7/+

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embryos. We found only basal transcription levels of
expression by qRT-PCR for these IC2-regulated genes in
DelTel7/+ placentae (Figure 1C). Wild type levels were
quite varied between samples, suggesting dynamic
changes in expression at this early developmental stage or
effects the genetic heterogeneity present in these crosses
on mixed background. Note the lack of spongiotropho-
blast and the expanded giant cell layer in the DelTel7/+
placenta (Figure 1B), consistent with what has been pub-
Figure 1 Expression of IC2-regulated genes in DelTel7/+ at E9.5. A) Diagram of distal mouse chromosome 7 (Chr 7) region showing the locations
of the imprinting centres 1 (IC1) next to H19 and 2 (IC2) in intron 10 of the Kcnq1 gene, as well as the protein-coding genes known to be regulated by
IC2 (Osbpl5, Phlda2, Cdkn1c, Kcnq1, Tssc4, Cd81, and Ascl2). This entire IC2-regulated cluster is deleted in the DelTel7 allele in which an array of telomere
repeats (Tel array) was introduced distal of Ins2 [36]. B) PHLDA2 immunohistochemistry (IHC) on sections of wild type (+/+) and maternal hemizygous
(DelTel7/+) placentae at E9.5. PHLDA2 IHC was performed on two placentae of each genotype. The PHLDA2 protein, localized to the labyrinth of the
wild type placenta, is absent in DelTel7/+ mutants. Note the lack of spongiotrophoblast and expanded giant cell layer in the DelTel7/+ placenta. Scale
bar: 1 mm. sp: spongiotrophoblast, lab: labyrinth, Gi: giant cells. C) qRT-PCR analysis of the imprinted genes Phlda2, Ascl2, and Cdkn1c, in E9.5 DelTel7/
+ placentae reveals virtually no expression from the intact paternal allele. Placentae were assessed at E9.5 before the embryonic lethality of the
DelTel7/+ observed at around E10.5. qRT-PCR was performed on five DelTel7/+ placentae and five wild type placentae with three technical replicates
per individual placentae. Expression is relative to the reference housekeeping gene, Peptidylprolyl isomerase A (Ppia). The error bar is the standard de-
viation from five biological replicates, assayed in triplicates. p < 0.02 for each qRT-PCR (t-test, unpaired, two-tailed).

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lished previously regarding the DelTel7/+ phenotype
[36].
Other then the entire IC2 sub-domain, DelTel7 also
deletes the very distal end of Chr7. This region contains
close to 20 annotated genes. Unlike genes in the IC1 and
IC2 domains, we find that most of these distal genes are
not expressed in the placenta at significant levels (Addi-
tional file 1). The absence of an obvious phenotype in
paternal hemizygotes for DelTel7 argues against haploin-
sufficiency effects in these mutants.
Germline transmission from DelTel7/IC2KO mice
IC2 is comprised of CpG-rich sequences within intron 10
of the Kcnq1 gene and functions in gene silencing, at least
in part, by serving as a promoter for the antisense ncRNA
Kcnq1ot1. We previously showed that paternal transmis-
sion of the IC2KO allele can rescue the embryonic lethal-
ity phenotype of DelTel7/+ embryos [36]. The rescued
DelTel7/IC2KO mice are missing the genes that are nor-
mally maternally expressed (they are deleted in DelTel7)
but expression of these genes is now supplied from the
paternal allele, since the IC2KO allele no longer produces
Kcnq1ot1 (Figure 2a) [33]. We confirmed the absence of
Kcnq1ot1 expression in DelTel7/IC2KO embryos and pla-
centae by strand-specific RT-PCR (Figure 2B). Pheno-
typic analysis of these compound heterozygous mice
therefore allows us to ask the following questions: (i) Are
all the maternally-expressed genes required for normal
placental development expressed from the IC2KO pater-
nal allele? (ii) Is the methylated IC2 required in the regu-
lation of maternally-expressed genes on the maternal
allele in wild type embryos?
We have previously shown that DelTel7/IC2KO pups sur-
vive to term, are recovered at the expected frequency and
are indistinguishable from their wild type litter mates at
birth (Oh et al, 2008). We now report that DelTel7/
IC2KO males and females are fertile and pass on the
mutant alleles at expected frequencies in their offspring.
DelTel7/IC2KO males crossed with wild type (+/+)
females produce litters of +/DelTel7 and +/IC2KO pups
at roughly equal frequencies (Table 1). DelTel7/IC2KO
females crossed with wild type males produce litters of
only the IC2KO/+ genotype as expected, since the
DelTel7/+ genotype is embryonic lethal (Table 1). Mice
produced from these crosses do not possess any obvious
developmental abnormalities and are able to survive to
adulthood. These results demonstrate that the mainte-
nance of imprinting is faithfully recapitulated in each
generation and that there are no aberrant effects of trans-
mitting either allele in the context of the compound
heterozygotes.
DelTel7/IC2KO placentae exhibit normal architecture
Because the DelTel7/+ genotype results in abnormal pla-
centation, we investigated the histology of several
DelTel7/IC2KO placentae compared to wild type litter
mates to determine whether there were any abnormalities
in placentation in the compound heterozygotes. Hema-
toxylin and eosin stain (H&E) revealed no apparent
abnormalities between DelTel7/IC2KO and wild type pla-
centae at E14.5, suggesting that the DelTel7/IC2KO pla-
centae displayed normal placental architecture (Figure
3A and 2B). To document this in greater detail, we used
in situ hybridization (ISH) and immunohistochemistry
(IHC) to analyze the distribution of the several lineage
markers in wild type and DelTel7/IC2KO placentae. Nei-
ther ISH for the placental lactogen-II gene Prl3b1 (Pl-II),
a mid- to late gestation marker of both the giant cell layer
Figure 2 Structure of the DelTel7 allele and absence of Kcnq1ot1 in rescued DelTel7/IC2KO embryos. A) Simplified representation of the struc-
ture of distal Chr7 in the DelTel7/IC2KO rescued animals, revealing the DelTel7 truncation on the maternal allele and the absence of IC2 and lack of
activation of Kcnq1ot1 expression on the paternal allele. M = maternal; P = paternal. C) RT-PCR demonstrating lack of Kcnq1ot1 expression in E9.5
DelTel7/IC2KO embryo and placenta.

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and mononucleated trophoblast cells of the labyrinth
(Figure 3C) [42], nor ISH for Tpbpa (4311), a marker of
the spongiotrophoblast layer (Figure 3D) [43] revealed
any overall differences in the these layers between the two
genotypes.
To determine whether there was a defect in the genera-
tion of the glycogen cell (GC) lineage in the DelTel7/
IC2KO placentae, both Periodic acid Schiff (PAS) stain
and ISH for Protocadherin-12 (Pcdh12), a gene specifi-
cally expressed in GCs [44] were performed (Figure 4A
and 4B). These analyses revealed no difference in the
overall number or distribution of GCs between the two
genotypes. For a detailed examination of the labyrinth,
we performed IHC for laminin, a marker of the basement
membrane in the labyrinth used for identifying fetal
blood vessels (Figure 4C) [45] and ISH for the imprinted
paternally expressed gene 1 (Peg1/Mest) which is also
expressed in the fetal endothelial cells that line the fetal
capillaries (Figure 4D). No abnormalities were observed
in the cell lineages analyzed or in the fetal capillaries of
the labyrinth of DelTel7/IC2KO placentae. Our results
show that DelTel7/IC2KO placentae are phenotypically
similar to wild type litter mates for the various represen-
tative markers analyzed.
To confirm the results of these marker analyses using an
independent method, we also performed expression anal-
ysis for Tpbpa (4311) and Peg1 qRT-PCR and observed
no difference between the two genotypes (Figure 5).
DelTel7/IC2KO placentae exhibit normal vasculature
Because we had no indication from our lineage marker
analysis that the DelTel7/IC2KO placentae had a drasti-
cally abnormal phenotype, we explored the possibility
that they may possess a subtle placental phenotype. One
of the most common subtle placental phenotypes is an
undervascularized labyrinth, otherwise known as a
"small" labyrinth [46]. Thus in addition to examining pla-
cental architecture, we also assessed whether the vascu-
larization of the DelTel7/IC2KO placentae was perturbed
using the vascular corrosion casting method [47]. We
found no apparent differences in the overall structure and
number of capillaries in the fetal vasculature of DelTel7/
IC2KO placentae compared to wild type litter mates at
E14.5 (Figure 6A-D). Moreover, morphometric analysis of
the average capillary diameter revealed no overall differ-
ence between DelTel7/IC2KO placentae and wild type
placentae (Figure 6E), with both having an average of
approximately 15 microns. This measure is in agreement
with the previously documented average capillary diame-
ter in wild type placentae [48].
DelTel7/IC2KO placentae exhibit proper expression of
imprinted genes
To determine whether DelTel7/IC2KO placentae express
the IC2-regulated genes from the paternal allele in a nor-
mal cell-type specific manner, we analyzed the expression
of three key genes in the distal Chr 7 region strongly asso-
ciated with placentation: Phlda2, Ascl2, and Cdkn1c (Fig-
ure 7). We chose to look at stages in which the expression
level of each of the genes is high or at its highest accord-
ing to our own observations as well as previously pub-
lished work [23,49,50]. IHC for Phlda2 on E10.5 DelTel7/
IC2KO placentae revealed exclusive expression in tro-
phoblast cells of the labyrinth, consistent with wild type
litter mates. ISH for Ascl2 on DelTel7/IC2KO placentae
revealed exclusive expression in cells of the spongiotro-
phoblast layer with some expression in glycogen cells
(GCs), consistent with wild type litter mates. ISH for
Cdkn1c on DelTel7/IC2KO placentae revealed high
expression in GCs and low expression throughout other
cell types in the labyrinth, as seen in wild type litter mates
(Figure 7). The relative expression level of each of these
genes was confirmed by quantitative RT-PCR (qRT-PCR).
No statistically significant differences in expression levels
between DelTel7/IC2KO and wild type placentae were
observed for Phlda2, Ascl2 and Cdkn1c at the stages ana-
lyzed (Figure 7A-C). Additionally, an examination of
expression level of several of the other imprinted genes in
the IC2 region (Tssc4, Kcnq1, and Cd81) by qRT-PCR at
E9.5 revealed no significant differences between DelTel7/
IC2KO and wild type placentae (Figure 7D).
Discussion
Placental abnormalities such as placentomegaly [41] and
placental mesenchymal dysplasia [40] are commonly
observed in mothers carrying BWS fetuses. Mouse mod-
els of the BWS imprinted region such as the Igf2 trans-
genic mouse model [51] and Cdkn1c null mouse models
[24-26] which accurately recapitulate several phenotypic
characteristics of the human disorder, are also accompa-
Table 1: Transmission of the DelTel7 ( ) and IC2KO alleles from compound heterozygotes
malefemaleprogeny
hetIC2KO het
+/+ /IC2KO0 12
/IC2KO+/+ 4037